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EP0718309A1 - Antigènes et anticorps pour la détection du collagène I - Google Patents

Antigènes et anticorps pour la détection du collagène I Download PDF

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Publication number
EP0718309A1
EP0718309A1 EP95120097A EP95120097A EP0718309A1 EP 0718309 A1 EP0718309 A1 EP 0718309A1 EP 95120097 A EP95120097 A EP 95120097A EP 95120097 A EP95120097 A EP 95120097A EP 0718309 A1 EP0718309 A1 EP 0718309A1
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Prior art keywords
peptide
gly
antibodies
collagen
amino acid
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EP95120097A
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German (de)
English (en)
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EP0718309B1 (fr
Inventor
Werner Dr. Naser
Brigitte Dr. Dräger
Ulrich Dr. Essig
Christa Dr. Hübner-Parajsz
Erasmus Dr. Huber
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Roche Diagnostics GmbH
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Roche Diagnostics GmbH
Boehringer Mannheim GmbH
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Priority claimed from DE1995103146 external-priority patent/DE19503146A1/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit

Definitions

  • the invention relates to antigens for the production of antibodies against N-termini of collagen I ⁇ 2 chains, a process for the production of such antigens, antibodies to N-termini of collagen I ⁇ 2 chains, which can be obtained by immunization with an antigen according to the invention, and the use such antibodies for the detection of collagen.
  • Collagen is an important structural protein in the connective tissue of skin, cartilage and bone.
  • 11 types are known, each consisting of three chains. Each type is composed of 3 different chains called ⁇ 1, ⁇ 2 and ⁇ 3 (E. Miller et al. In Methods in Enzymology 144, Structural and Contractile Proteins, ed. L. Cunningham, Academic Press Inc. 1987, p 3 - 41).
  • a characteristic property of the mature collagen of certain tissues, such as in particular bones or cartilage, is the crosslinking of fibers lying next to one another by means of hydroxylysylpyridinoline or lysylpyridinoline (Fujimoto, D., et al., (1978) J.
  • a disadvantage of these peptides isolated from natural sources is that there is no reliable source for reproducible production of the antigens or binding partners in the test.
  • Another disadvantage of the peptides isolated from natural sources is the risk of contamination with infectious material.
  • Defined antigens can e.g. can be obtained by chemical synthesis of a peptide which corresponds to an epitope of the antigen. If small peptides with a molecular weight of about 700 - 1500 D are used for this purpose, binding to a carrier molecule is necessary in order to obtain an antigen with an immunogenic effect. By binding to the carrier molecule, the structure of the epitope must not be changed.
  • the coupling to the carrier molecule has therefore preferably been carried out at the ends of the peptide chain at a sufficient distance from the suspected epitope area (Laboratory Technics in Biochemistry and Molecular Biology, Synthetic Polypeptides as Antigens, Editors RH Burdon and PH van Knippenberg, Elsevier, Amsterdam, New York, Oxford 1988, pages 95-100).
  • a problem in the chemical synthesis of a defined antigen which corresponds to a natural degradation product of crosslinked collagen is the hydroxylysyl or lysylpyridinoline structure resulting from the crosslinking, the chemical synthesis of which is complex.
  • WO 94/03813 also describes synthetic linear peptides, which correspond to a sequence of the non-helical C- or N-terminal region of collagen, as antigens for the production of antibodies against collagen or collagen fragments. All contiguous amino acid sequences of the non-helical C- or N-terminal region of collagen are suitable as synthetic linear peptides. These ranges are from Chuetal., Nature 310, 337-340 (1984), Click et al., Biochemistry 9, 4699-4706 (1970), Morgan et al., J. Biol. Chem. 245, 5042-5048 (1970 ) and Bernard et al., Biochemistry 22, 5213-5223 (1983). In particular, peptides from the C-terminal region of the ⁇ 1 chain of collagen have been disclosed. The antibodies obtainable with these immunogens can be used in immunoassays for the detection of collagen breakdown products in body fluids.
  • the object of the present invention was to further improve this type of immunoassay and to further optimize the necessary input materials such as antibodies and antigens.
  • the antibodies must be characterized by the fact that they contain the collagen breakdown products that occur in body fluids, i.e. Specifically recognize collagen fragments.
  • the present invention therefore relates to an antibody against collagen breakdown products from collagen I in body fluids, which specifically recognizes the amino acid sequence Val-Gly-Leu-Gly within collagen I or fragments thereof.
  • This amino acid sequence corresponds to amino acid residues 7 to 10 of the N-terminal end of the collagen type I ⁇ 2 chain. It has surprisingly been found that an immunoassay for the detection of collagen breakdown products when using these antibodies can firstly detect the collagen breakdown products in the body fluids very specifically and sensitively and that such an immunoassay has a very good correlation to an immunoassay which uses antibodies against crosslinked collagen breakdown products ( WO 89/04491).
  • the antibodies according to the invention specifically recognize the amino acid sequences Val-Gly-Leu-Gly. This means that this amino acid sequence is recognized within the collagen breakdown products that occur in the body fluids.
  • the amino acids adjacent to this sequence can also be recognized by the antibodies. However, this is not a condition for the antibodies according to the invention, since these amino acids may also be missing in some collagen fragments. It is crucial that the antibodies recognize the epitope characterized by the sequence Val-Gly-Leu-Gly within a collagen fragment.
  • the total epitope bound by the antibody can be a few amino acids longer, for example 1-4 amino acids.
  • Particularly preferred antibodies recognize the amino acid sequence 6-13 of the N-terminal end of the collagen type I ⁇ 2 chain.
  • the antibodies can be produced by commonly used methods. For example, collagen fragments which contain the N-terminal type I ⁇ 2 chain and which contain the sequence Val-Gly-Leu-Gly can be used for immunization from body fluids. These collagen fragments can also be produced by enzymatic or chemical cleavage of type I collagen. The antibodies that are formed against these more or less strongly defined peptides must be selected or purified after the immunization using a suitable screening method. The reaction of the antibodies against short peptides, which contain the sequence Val-Gly-Leu-Gly, is tested and used.
  • the production of the antibodies by immunizing the animals with a synthetic peptide or peptide derivative which has an amino acid sequence corresponding to SEQ ID NO 1 or a partial sequence thereof which comprises at least the amino acid sequence Val-Gly-Leu-Gly is considerably simpler and therefore particularly preferred. includes.
  • the synthetic peptide preferably contains at least 6 amino acids.
  • the amino acids N- or / and C-terminal of this sequence are preferably added, which are also adjacent to this sequence in the type I ⁇ 2 collagen chain.
  • other amino acids can also be added.
  • the peptides are coupled to an immunogenic carrier, for example keyhole limpet hemocyanins (KLH), bovine serum albumin or edestin, in order to increase the immune response.
  • an immunogenic carrier for example keyhole limpet hemocyanins (KLH), bovine serum albumin or edestin, in order to increase the immune response.
  • KLH keyhole limpet hemocyanins
  • bovine serum albumin bovine serum albumin
  • edestin edestin
  • the antibodies can be coupled with a signal-generating substance or labeling group, for example fluorescent labeling groups, (electro) chemiluminescent labeling groups, digoxin, biotin or radioactive groups.
  • a signal-generating substance or labeling group for example fluorescent labeling groups, (electro) chemiluminescent labeling groups, digoxin, biotin or radioactive groups.
  • the antibodies can be coupled to them directly or indirectly. These groups or the solid phase are coupled by the methods known to the person skilled in the art.
  • the invention further relates to peptides or peptide derivatives which contain the amino acid sequence corresponding to SEQ ID NO 1 or a partial sequence thereof which has at least the amino acid sequence Val-Gly-Leu-Gly.
  • the amino acid sequences are partial regions from the N-terminal non-helical ends of the collagen type I ⁇ 2 chain.
  • the Peptides or peptide derivatives can be used as immunogens for the production of the antibodies according to the invention, as specific binding partners and as standard material in immunoassays.
  • the peptides according to the invention can be generated by known synthetic methods by means of chemical methods, or can be produced by genetic engineering in a suitable host cell by cloning and expressing a DNA sequence coding for these peptides.
  • the invention also relates to peptide derivatives.
  • This term encompasses peptides in which one or more amino acids have been derivatized by a chemical reaction. Examples are in particular those molecules in which the backbone and / or free amino acid groups have been derivatized.
  • the peptide derivatives according to the invention have an essentially equivalent specificity and / or affinity for binding to the antibodies according to the invention.
  • Peptide derivatives are also understood to mean peptide-mimetic substances which have an essentially equivalent specificity and / or affinity for binding to the antibodies according to the invention, such as the aforementioned peptides.
  • Peptide-mimetic substances are compounds which can replace peptides in their interaction with the antibodies and can have increased stability compared to the native peptides. Methods for the production are described by Giannis and Kolter, Angew. Chem. 105 (1993), 1303-1326, Lee et al., Bull. Chem. Soc. Jpn. 66 (1993), 2006-2010 and Dorsch et al., Contacts (Darmstadt) (1993) (2), 48-56.
  • the peptides or peptide derivatives according to the invention can, as already described above, signal-generating substances or labeling groups on immunogenic carriers, e.g. Fluorescent labeling groups, digoxin, biotin or radioactive groups can be coupled.
  • signal-generating substances or labeling groups on immunogenic carriers e.g. Fluorescent labeling groups, digoxin, biotin or radioactive groups
  • the peptides or peptide derivatives can be bound directly or indirectly to this solid phase, for example tubes, spheres, latex particles, etc., for example via a streptavidin / biotin interaction. The coupling takes place according to common procedures.
  • these peptides or peptide derivatives according to the invention can be used for the production of antibodies.
  • Another object of the invention is a method for producing antibodies against collagen breakdown products of collagen by immunization with a peptide or peptide derivative according to the invention, which has the amino acid sequence corresponding to SEQ ID NO 1 or a partial sequence thereof, which has at least the amino acid sequence Val-Gly-Leu-Gly , includes.
  • the peptide or peptide derivative preferably has a length of at least 6 amino acids and is coupled to an immunogenic carrier. After the immunization has taken place, the desired antibodies are isolated from the serum of the immunized animals by known methods.
  • the desired antibody is preferably isolated by immunoadsorption on a peptide coupled to a carrier, preferably Sepharose, which contains the amino acid sequence corresponding to SEQ ID NO 1 or a partial sequence thereof which has at least the amino acid sequence Val-Gly-Leu-Gly.
  • a carrier preferably Sepharose
  • a preferred object of the invention is a process for the production of monoclonal antibodies against cross-linked collagen by immunization with a peptide or peptide derivative according to the invention, immortalization of the spleen cells of the immunized animals, cloning of those immortalized spleen cells which produce the desired antibody and isolation of the antibody from the cloned Cells or the culture supernatant of these cells.
  • the immunization takes place in the animals usually used for this purpose, preferably mice or rabbits.
  • the spleen cells of the immunized animals are immortalized according to methods familiar to the person skilled in the art, e.g. hybridoma technology (Köhler and Milstein, Nature 256 (1975), 495-497) or by transformation with the Epstein-Barr virus (EBV transformation).
  • hybridoma technology Keröhler and Milstein, Nature 256 (1975), 495-497
  • EBV transformation Epstein-Barr virus
  • a sample of the culture supernatant is incubated in a conventional immunoassay with the peptide or peptide derivative according to the invention used for immunization and it is examined whether an antibody binds to this peptide or peptide derivative.
  • the concentration of collagen breakdown products is an important diagnostic marker for the extent of osteolysis.
  • the concentration of collagen breakdown products is an important diagnostic marker for the extent of osteolysis.
  • Another object of the invention is therefore the use of a polyclonal or monoclonal antibody according to the invention for determining osteolysis by incubating the antibody with a sample and determining the collagen breakdown products binding to the antibody or an immunoassay for the detection of collagen I or collagen fragments in a sample Antibody according to the invention is used.
  • a body fluid in which collagen breakdown products can be located preferably serum, plasma or urine, is used as the sample.
  • the binding of the antibody to collagen I or collagen fragments is preferably determined by means of a competitive test.
  • the antibodies can be incubated with the sample and with the additionally contained peptide or peptide derivative according to the invention both simultaneously and sequentially.
  • the collagen fragments contained in the sample preferably compete with the peptide or peptide derivative according to the invention for binding to the antibody, so that the binding of the antibody to the hapten according to the invention represents a measure of the amount of antigen in the sample.
  • a heterogeneous competitive immunoassay in which the liquid phase is separated from the solid phase, both the antibody or the peptide can be labeled or bound to the solid phase. The exact amount of antigen in the sample can then be determined in the usual way by comparison with a standard treated in the same way.
  • TINIA turbidimetic inhibition immunoassay
  • LPIA latex particle immunoassay
  • TINIA the peptide or peptide derivative according to the invention is bound to a carrier such as dextran (EP-A-0 545 350).
  • DExtran EP-A-0 545 350
  • This polyhapten competes with the analyte contained in the sample for binding to the antibody.
  • the complex formed can be determined turbidimetrically or nephelometrically.
  • LPIA particles preferably latex particles
  • LPIA particles are coated with the peptides according to the invention and mixed with the antibody according to the invention and the sample. Agglutination is reduced when the analyte is present in the sample.
  • Enzyme immunoassays (Wisdom, GB, Clin. Chem. 22/8 (1976), 1243-1255 and M. Oellerich, J. Clin. Chem. Clin. Biochem. Vol 18 (1980), 197 - 208), fluorescence polarization immunoassays (FPIA) (W. Dandliker et al., J. Exp. Med. 122 (1965), 1029), the enzyme multiplied immunoassay technique (EMIT) (Gunzer et al., Contacts III, 1980, 3-11 and K. Rubenstein, Biochemical and Biophysical Research Communications 47 (1972), 846-851) or CEDIA technology (Henderson et al., Clinical Chemistry 32 (1986), 1637-41).
  • the sample is pretreated with the denaturant for proteins known to those skilled in the art prior to incubation with the antibody.
  • the denatured sample is preferably diluted with the antibody before incubation. All agents known to the person skilled in the art are suitable as denaturing agents for this.
  • KSCN Potassium rhodanide
  • TTAB tetradecyltriethylammonium bromide
  • Another object of the invention is a standard material for creating a standard or calibration curve in an immunoassay for the detection of collagen I or collagen fragments, characterized in that it contains a peptide or peptide derivative which has the amino acid sequence corresponding to SEQ ID NO 1 or a partial sequence therefrom, which has at least the amino acid sequence Val-Gly-Leu-Gly.
  • SEQ ID NO. 1 shows the sequence of a peptide according to the invention which corresponds to amino acids 1-13 of the N-terminal end of the collagen type I ⁇ 2 chain.
  • Xaa means Gln or Pyr (pyroglutamine).
  • Fmoc fluorenylmethyloxycarbonyl
  • the amino acids or amino acid derivatives are dissolved in N-methylpyrrolidone.
  • the peptide is synthesized on 3 g of 4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy resin (Tetrahedron Letters 28 (1987), 2107) with a loading of 0.87 mmol / g (JACS 95 ( 1973), 1328).
  • the coupling reactions are carried out with respect to the Fmoc amino acid derivative using 4.4 equivalents of dicylcohexylcarbodiimide and 4.8 equivalents of N-hydroxybenzotriazole in dimethylformamide as the reaction medium for 60 minutes.
  • the coupling success is checked by means of the Kaiser test (Anal. Biochem.
  • the peptide is released from the synthetic resin and the acid-labile protective groups are split off with 80 ml of trifluoroacetic acid, 5 ml of ethanedithiol, 2.5 g of phenol, 2.5 ml of m-cresol and 5 ml of water in 60 minutes at room temperature.
  • the reaction solution is then concentrated in vacuo, the residue is taken up in diisopropyl ether, stirred vigorously for 1 / 2-2 hours and then filtered off.
  • the material is then pre-cleaned by means of gel permeation chromatography on Sephadex G15, using 0.5% acetic acid as the eluent.
  • the raw material obtained is then filtered off and prepared by means of a preparative HPLC on Nucleosil RP18 (column 40 mm ⁇ 250 mm 300 ⁇ , 5 ⁇ m) over a gradient of 100% reagent A (water, 0.1%, trifluoroacetic acid) to 100% reagent B. (60% acetonitrile, 40% water, 0.1% trifluoroacetic acid) isolated in 120 minutes. The identity of the eluted material is checked by means of ion spray mass spectrometry.
  • the peptides were prepared using Fmoc (fluorenyloxycarbonyl) solid phase synthesis. The reactions were carried out on a multiple synthesizer from Zinsser (SMP 360) peptide synthesizer. The coupling reactions were carried out with respect to the Fmoc-amino acid derivative with 1.1 equivalents of dicyclohexylcarbodiimide and 1.0 equivalents of N-hydroxybenzotriazole over 90 minutes. Dimethylformamide was used as the reaction medium. The Fmoc group was split off using 50% piperidine in DMF in 10 and 20 minutes. 10.0 equivalents of the following Fmoc-amino acid derivatives were used: Asp (with tert.
  • the peptide was synthesized on 20 mg Rink resin (polystyrene / 1% divinylbenzene) with a charge of 0.50 mmol / g.
  • SEQ. ID NO: 2 Gln, Tyr, Asp, Gly, Lys, Gly, Val, Gly, Leu, Gly, Pro, Met, Gly, Leu, Met, Gly, Pro, Arg, Gly.
  • the peptide is released with 1 ml trifluoroacetic acid, 50 ⁇ l ethanedithiol, 50 ⁇ l thio-cresol, 25 ⁇ l thioanisole and 25 ⁇ l water in 3 hours at room temperature.
  • the peptide was precipitated from the cleavage solution with diisopropyl ether.
  • mice Eight days after the fourth immunization, retroorbital blood was drawn to obtain mouse serum for analytical evaluation. Immunization results with the peptide coupled to KLH (CollI ⁇ 2 (1-13, Gln / Pyr) -Cys-KLH) were better compared to the RSA carrier protein. For further investigation, the 2 mouse sera 328/14 and 328/11 were characterized in more detail from the 15 mice which were immunized with the KLH immunogen (see Example 3.1).
  • mice were killed by a broken neck and the spleens were removed sterile.
  • the spleen cells were expressed in RPMI 1640 base medium using forceps from the connective tissue envelope.
  • the cell suspension was rubbed through a steel sieve and then centrifuged in a centrifuge tube in RPMI 1640 base medium at 200 g.
  • Spleen cells from an immunized mouse were mixed with P3x63 Ag8-653 myeloma cells (ATTCC-CRL 8375) in a ratio of 1: 5 and centrifuged (10 minutes, 300 g, 4 ° C.). The cells were washed once more with RPMI 1640 base medium and centrifuged at 400 g in a 50 ml pointed tube. The supernatant was dumped, the cell sediment was loosened, 1 ml of PEG (MG 4000, Merck) was added and pipetted through.
  • PEG MG 4000, Merck
  • Microtiter plates (from Nunc) were coated with thermal RSA streptavidin and loaded with a solution of 100 ng of the respective biotinylated peptides per ml of PBS, 0.05% Tween 20 (100 ⁇ l / well).
  • the following peptides are involved: Coll I ⁇ 2 (1-13, 1 Gln), Coll I ⁇ 2 (1-13, 1Pyr), a 1: 1 mixture of these two peptides Coll I ⁇ 2 (1-13, 1Gln, Pyr), Coll I ⁇ 2 (1 -8), Coll I ⁇ 2 (2-9) ... to Coll I ⁇ 2 (14-21).
  • mice sera 328/14 and 328/11 were diluted 1: 4000 in PBS, 0.05% Tween 20, 100 ⁇ l of these solutions were then pipetted into the wells coated with the peptides and then incubated for 1 hour at room temperature. The mixture was then washed 3 times with PBS, 0.05% Tween 20.
  • 100 ⁇ l of a POD-labeled Fab fragment of a polyclonal antibody from sheep against mouse Fc- ⁇ Boehringer Mannheim, Ident No.
  • an enzyme immunoassay can be set up according to the competition principle for measuring collagen peptides in urine, which has a good correlation to the Osteomark® test from the company Ostex.
  • Thermo-RSA-streptavidin-coated microtiter plates were coated with the biotinylated peptide mixture Coll I ⁇ 2 (1-13, 1 Gln / Pyr, 1: 1 mixture) (100 ng / ml PBS, 0.05% Tween 20, thereof 100 ⁇ l / well ). After incubation at room temperature for one hour, the mixture was washed twice with PBS, 0.05% Tween 20. Dilutions with PBS, 0.05% Tween 20 were prepared from the peptide mixture Coll I ⁇ 2 (1-13, 1 Gln / Pyr) in the following concentration ranges: 0, 25, 50, 100, 250, 500 and 1000 ng / ml.
  • mice serum dilution 50 ⁇ l of the respective standard and 50 ⁇ l of the mouse serum dilution (mouse serum 328/11, diluted 1: 4000 in PBS, 0.05% Tween 20) were pipetted into each well and mixed with one another. After incubation at room temperature for 1 hour, the mixture was washed 3 times with PBS, 0.05% Tween 20 and then, as described in the example of the Pepscan measurements, with Fab-labeled POD-labeled Fab fragment of a polyclonal antibody against mouse Fc ⁇ and subsequent color reaction, the measurement signal was generated. The plot of the measurement signal against the standard concentration gives the standard curve (Fig. 2).
  • mice 328/11 and 328/14 fused with myeloma cells were sown.
  • a pepscan analysis was carried out with the culture supernatants 9/1 to 9/12 (mouse 328/11) and 10/1 to 10/4 (mouse 328/14). The results are shown in Figs. 4 and 5.
  • test procedure corresponds to Example 3.1 with the following variants:
  • Thermo-RSA-streptavidin-coated microtiter plates were coated with the biotinylated peptide Coll I ⁇ 2 (6-13) (100 ng / ml PBS, 0.05% Tween 20, of which 100 ⁇ l / well). After incubation at room temperature for one hour, the mixture was washed twice with PBS, 0.05% Tween 20. The following concentrations were prepared from the peptide mixture Coll I ⁇ 2 (1-13, 1 Gln / Pyr) by dilution with PBS, 0.05% Tween 20: 0, 50, 100, 200 ng / ml.
  • Table 3 summarizes data for further culture supernatants. In all cases there was a very good correlation to the Osteomark® test. Table 3 KÜ Verd. 0 hours in my opinion 200 ng / ml ME Number of urines Correlation to Osteomark® r 9-1 1 + 1 1663 615 6 0.972 9-3 1 + 2 1391 397 5 0.995 9-4 1 + 2 1536 334 5 0.964 9-8 1 + 1 953 222 5 0.977 9-9 1 + 59 1349 341 8th 0.925 10-2 undiluted 461 114 4th 0.949 ku: culture supernatant Verd .: Dilution 0 hours: lowest standard 200 ng / ml: highest standard r: correlation coefficient

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EP95120097A 1994-12-23 1995-12-19 Antigènes et anticorps pour la détection du collagène I Expired - Lifetime EP0718309B1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE4446232 1994-12-23
DE4446232 1994-12-23
DE1995103146 DE19503146A1 (de) 1995-02-01 1995-02-01 Antigene und Antikörper zum Nachweis von Kollagen I
DE19503146 1995-02-01

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EP0718309A1 true EP0718309A1 (fr) 1996-06-26
EP0718309B1 EP0718309B1 (fr) 1999-04-07

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JP (1) JPH08233811A (fr)
DE (1) DE59505585D1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998026286A2 (fr) * 1996-12-09 1998-06-18 Osteometer Biotech A/S Dosages en sandwich de fragments de type i de collagene
US5817755A (en) * 1995-05-19 1998-10-06 Washington Research Foundation Synthetic peptide analogs of cross-linked N-telopeptides of type I collagen
US6255056B1 (en) 1998-06-19 2001-07-03 Washington Research Foundation Cartilage resorption assays
US6300083B1 (en) 1996-08-22 2001-10-09 Osteometer Biotech A/S Assaying D-amino acids in body fluids
US6348320B1 (en) 1998-06-19 2002-02-19 Washington Research Foundation Cartilage resorption assays measuring type II collagen fragments
US6602980B1 (en) 1998-06-19 2003-08-05 Washington Research Foundation Collagen type III synthetic peptides for collagen resorption assays
US6916903B2 (en) 1998-06-19 2005-07-12 Washington Research Foundation Collagen type III synthetic peptides for collagen resorption assays

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9506050D0 (en) 1995-03-24 1995-05-10 Osteometer A S Assaying collagen fragments in body fluids
WO1996012193A1 (fr) 1994-10-17 1996-04-25 Osteometer Bio Tech A/S Evaluation du schema de fragmentation du collagene dans des fluides corporels et diagnostic des troubles associes au metabolisme du collagene
US6107047A (en) * 1996-03-21 2000-08-22 Osteometer Biotech A/S Assaying protein fragments in body fluids
WO2009051281A1 (fr) * 2007-10-19 2009-04-23 Tonen Chemical Corporation Extrudeuse possédant deux étages de mélange et procédé servant à préparer un mélange de polymère et de diluant
DK2770327T3 (en) * 2009-03-30 2017-08-28 Nordic Bioscience As BIOMARKERING ASSAY FOR FIBROSE
JPWO2023068248A1 (fr) * 2021-10-20 2023-04-27
EP4502602A1 (fr) * 2022-03-25 2025-02-05 Sekisui Medical Co., Ltd. Procédé de détection immunologique et kit de détection immunologique

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Publication number Priority date Publication date Assignee Title
WO1992021698A1 (fr) * 1991-05-31 1992-12-10 Washington Research Foundation DETECTION DE LA DEGRADATION DU COLLAGENE $i(IN VIVO)
DE4225038A1 (de) * 1992-07-29 1994-02-03 Boehringer Mannheim Gmbh Herstellung und Verwendung von Antikörpern gegen Kollagen
EP0699752A2 (fr) * 1994-07-22 1996-03-06 Collagen Corporation Collagène recombinant muté

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992021698A1 (fr) * 1991-05-31 1992-12-10 Washington Research Foundation DETECTION DE LA DEGRADATION DU COLLAGENE $i(IN VIVO)
DE4225038A1 (de) * 1992-07-29 1994-02-03 Boehringer Mannheim Gmbh Herstellung und Verwendung von Antikörpern gegen Kollagen
EP0699752A2 (fr) * 1994-07-22 1996-03-06 Collagen Corporation Collagène recombinant muté

Non-Patent Citations (1)

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Title
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5817755A (en) * 1995-05-19 1998-10-06 Washington Research Foundation Synthetic peptide analogs of cross-linked N-telopeptides of type I collagen
US6300083B1 (en) 1996-08-22 2001-10-09 Osteometer Biotech A/S Assaying D-amino acids in body fluids
WO1998026286A2 (fr) * 1996-12-09 1998-06-18 Osteometer Biotech A/S Dosages en sandwich de fragments de type i de collagene
WO1998026286A3 (fr) * 1996-12-09 1998-08-13 Osteometer Biotech As Dosages en sandwich de fragments de type i de collagene
US6660481B2 (en) 1996-12-09 2003-12-09 Osteometer Biotech A/S Sandwich assays for collagen type I fragments
US6255056B1 (en) 1998-06-19 2001-07-03 Washington Research Foundation Cartilage resorption assays
US6348320B1 (en) 1998-06-19 2002-02-19 Washington Research Foundation Cartilage resorption assays measuring type II collagen fragments
US6566492B2 (en) 1998-06-19 2003-05-20 Washington Research Foundation Synthetic peptides of type II collagen for cartilage resorption assays
US6602980B1 (en) 1998-06-19 2003-08-05 Washington Research Foundation Collagen type III synthetic peptides for collagen resorption assays
US6916903B2 (en) 1998-06-19 2005-07-12 Washington Research Foundation Collagen type III synthetic peptides for collagen resorption assays

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US5821065A (en) 1998-10-13

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